There is a critical need to better understand the enzootic transmission cycles of West Nile virus (WNV), particularly as relates to understanding the role that host genes play in determining why different species and strains of mosquito vary in their susceptibility to WNV infection and how such variation impacts disease transmission. The long term goal of this study is to understand how the molecular interactions that occur between arboviruses and their hosts determine infection susceptibility. A major current impediment to the systematic identification of mosquito host genes that are important in WNV infection is the impracticality of doing functional genetic screens in mosquitoes. The objective of this project is to evaluate the utility of using unbiased, functional genetic screens in Drosophila melanogaster as an alternative experimental approach to identifying mutations in host genes important for WNV infection. This objective is predicated on the hypothesis that using D. melanogaster as a surrogate host for WNV infection will provide a genetically tractable model system to expedite the discovery of host genes important for infection. This hypothesis is based on preliminary studies demonstrating that D. melanogaster can be infected by WNV. Successful utilization of D. melanogaster to identify host genes important for WNV infection would provide an empirically based starting point for identifying mosquito genes that modulate infection susceptibility in important mosquito vectors of the virus. One specific aim is proposed: identify mutations in D. melanogaster that affect the ability of WNV to infect flies. This aim is based on the working hypothesis that mutations in D. melanogaster genes involved in WNV infection will lead to infections that have readily detectable alterations in virus titer. Two genetic screens are proposed in which EMS-induced recessive mutations or P element-induced misexpression mutations will be screened for their effects on WNV infection by injecting virus into flies containing either mutagenized chromosomes or genome-wide P-element insertions that induce the misexpression of neighboring genes and quantifying the production of WNV genomic RNA by RT-PCR. The proposed studies are significant, because they may provide a proof-of- principle demonstration that the powerful genetic methodologies available in D. melanogaster can be used to overcome technical limitations presented by natural mosquito hosts of WNV in order to identify host genes important for WNV infection. Public Health Relevance: Having a genetically tractable model system to investigate host:arbovirus interactions would greatly facilitate the identification of host genes important for WNV infection and would provide the tools for the elucidation of how those genes impact enzootic transmission cycles and, ultimately, disease transmission. Until such host genes have been identified, a full understanding of host:WNV interactions that are important for determining infection susceptibility will remain limited, impeding efforts to develop effective strategies for reducing the health burden caused by the emergence and spread of WNV disease.